Estrogenic compounds, methods of using and methods of administering the same

ABSTRACT

Novel estrogenic compounds of Formula I are provided. 
                         
wherein the bond represented by the wavy line may be a single or double bond such that when the wavy line is a single bond, R 1  is selected from the group consisting of hydrogen, sulfate and glucoronate or other esters, and when the wavy line is a double bond, R 1  does not exist; R 2  is lower alkyl; R 3  may be selected from the group consisting of hydrogen, sulfate, or glucuronide or other esters; and R 4  through R 13  may independently be selected from the group consisting of hydrogen, hydroxy, ketone, lower alkyl, lower alkoxy, halogen, and carbonyl groups and R 14  is selected from the group consisting of hydrogen, sulfate and glucoronide and other esters. When R 1  is hydroxy, the hydroxy or ester substituent may have either an α or a β orientation. Compositions of matter including compounds of the present invention are also provided as are methods of treating mammals in need of treatment using compounds of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 11/671,718, filed Feb. 6, 2007, now U.S. Pat. No.7,459,445, which is a continuation application of U.S. patentapplication Ser. No. 10/628,057, filed Jul. 23, 2003, now U.S. Pat. No.7,179,799, which claims priority to U.S. patent application Ser. No.09/800,614, filed Mar. 8, 2001, now U.S. Pat. No. 6,660,726, which inturn, claims priority to U.S. Provisional Application Ser. No.60/188,523, filed Mar. 10, 2000, the disclosures of each of which arehereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the isolation of estrogenic compounds.

BACKGROUND OF THE INVENTION

Women, particularly menopausal and post-menopausal women, oftenexperience a wide variety of conditions and disorders attributable toestrogen deprivation. Estrogen deprivation is most often the result ofloss of ovarian function. Exemplary conditions are hot flashes, drynessof the vagina, including discomfort during intercourse, loss of bonemass, increased heart disease and the like.

Providing dosages of estrogen is an effective agent for the control orprevention of such conditions, particularly in controlling or preventinghot flashes and vaginal atrophy, along with retarding or preventingosteoporosis. Estrogen is typically administered alone or in combinationwith a progestin.

As detailed in U.S. Pat. No. Re. 36,247 to Plunkett et al., estrogenalone, given in small doses, on a continuous basis, is effective in mostpatients for the control of the above symptoms and problems associatedtherewith. However, although the vast majority of women takingcontinuous low-dose estrogen will not have bleeding for many months oreven years, there is a distinct risk posed by this routine of silently(i.e. exhibiting no overt symptoms) developing “hyperplasia of theendometrium”. This term refers, of course, to an overstimulation of thelining of the uterus which can become pre-malignant, coupled with thepossibility that the patient may eventually develop cancer of theuterine lining even under such a low-dose regimen (Gusberg et al.,Obstetrics and Gynaecology, 17, 397-412, 1961).

Estrogen alone can also be given in cycles, usually 21-25 days ontreatment and 5-7 days off treatment. Again, if small doses of estrogenare required to control the symptoms and it is used to this fashion,only about 10% of women will experience withdrawal bleeding between thecycles of actual treatment. However, one must again be concerned by therisk of developing endometrial hyperplasia and by the increased relativerisk of developing cancer of the uterus (Research on the Menopause:Report of a W.H.O. Scientific Group, 53-68, 1981).

The addition of progestin for the last 7-10 days of each estrogen cyclemay virtually eliminate the concern about developing endometrialhyperplasia and/or also reduce the risk of developing endometrialcarcinoma below that of the untreated general population. However,withdrawal bleeding may occur regularly in this routine and this ishighly unacceptable to most older women (Whitehead, Am. J. Obs/Gyn.,142, 6, 791-795, 1982).

Still another routine for estrogen administration may involve aformulation such as those found in birth control pills which containrelatively small doses of estrogen over the full 20-21 day treatmentcycle, plus very substantial doses of potent progestins over the sameperiod of time. This routine, of course not only produces withdrawalbleeding on each cycle, but is further unacceptable because suchformulations have been shown to carry an increased risk of developingarterial complications, such as stroke or myocardial infarction in olderwomen about the age of 35-40. This is especially true if the individualis a smoker of cigarettes (Plunkett, Am. J. Obs/Gyn. 142, 6, 747-751,1982). There, however, remains a need for novel isolated estrogeniccompounds.

SUMMARY OF THE INVENTION

Thus, as one aspect of the present invention, a compound represented byFormula I is provided.

where the bond represented by the wavy line may be a single or doublebond such that when the wavy line is a single bond, R₁ may be selectedfrom the group consisting of hydrogen, sulfate and glucoronide or otheresters, and when the wavy line is a double bond, R₁ does not exist; R₂is lower alkyl; R₃ may be selected from the group consisting ofhydrogen, sulfate, and glucuronide or other esters; and R₄ through R₁₃may independently be selected from the group consisting of hydrogen,hydroxy, ketone, lower alkyl (C₁ to C₄), lower alkoxy (C₁ to C₄),halogen, and carbonyl groups. When R₁ is hydroxy, the hydroxy or estersubstituent may have either an α or a β orientation, with the βorientation being preferred. R₂ is preferably C₁ to C₄ alkyl, and morepreferably is methyl. R₄ through R₁₂ are preferably hydrogen. R₁₃ ispreferably hydrogen or ethynyl. R₁₄ is hydrogen, sulfate, or glucoronideand other esters.

The compound represented by Formula I may be present in chemically pureform, namely greater than about 90% pure, preferably greater than about95% pure, and most preferably greater than about 99% pure.

A preferred compound is illustrated in Formula II:

Another preferred compound is illustrated in Formula III:

In another aspect, the present invention provides a composition ofmatter. The composition of matter comprises a compound according to thepresent invention.

In still another aspect, the invention provides a method of treatingmammals in need of treatment. The method comprises administering aneffective amount of a composition of matter according to the presentinvention. Examples of treatments that are addressed by the compositionsof the invention include vasomotor symptoms, atrophic vaginitis, andosteoporosis.

The invention is described in greater detail with respect to thepreferred embodiments set forth hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings which are incorporated in and constitute apart of the specification, illustrate embodiments of the invention and,together with the description, serve to explain principles of theinvention.

FIG. 1 is a HPLC Chromatogram using chromatographic method 1 showingPeaks A and B in the Endeavor 10-Component Conjugated Estrogens drugproduct;

FIG. 2 is a HPLC Chromatogram using chromatographic method 1 showingPeak A in the 17α-dihydroequilin sulfate, sodium salt standard;

FIG. 3 is a HPLC Chromatogram using chromatographic method 1 showingPeak B in the Equilin sulfate, sodium salt standard;

FIG. 4 is a HPLC Chromatogram using chromatographic method 4 showingre-injection of Peak A to determine its approximate purity;

FIG. 5 is a low resolution negative ion FAB-MS spectrum of Peak A;

FIG. 6 is a full range 400 MHz ¹H-NMR spectrum of Peak A in d₆-DMSO;

FIG. 7 is a 400 MHz ¹H-NMR spectrum of the aliphatic region of Peak A ind₆-DMSO;

FIG. 8 is a 400 MHz ¹H-NMR spectrum of the aromatic region of Peak A ind₆-DMSO;

FIG. 9 is a full range 400 MHz 2D COSY ¹H-NMR spectrum of Peak A ind₆-DMSO;

FIG. 10 is a 400 MHz 2D COSY ¹H-NMR spectrum of the aliphatic region ofPeak A in d₆-DMSO;

FIG. 11 is a 400 MHz 2D COSY ¹H-NMR spectrum of the aromatic region ofPeak A in d₆-DMSO;

FIG. 12 is a full range 100 MHz ¹³C-NMR spectrum of Peak A in d₆-DMSO;

FIG. 13 is a 100 MHz ¹³C-NMR spectrum of the aliphatic region (0-40 ppm)of Peak A in d₆-DMSO;

FIG. 14 is a 100 MHz ¹³C-NMR spectrum of the aliphatic region (40-80ppm) of Peak A in d₆-DMSO;

FIG. 15 is a 100 MHz ¹³C-NMR spectrum of the aromatic region of Peak Ain d₆-DMSO;

FIG. 16 is a full range 2D HMQC spectrum of the correlations of theprotons and carbons of Peak A in d₆-DMSO;

FIG. 17 is a 2D HMQC spectrum of the correlations of the aliphaticprotons and carbons in Peak A in d₆-DMSO;

FIG. 18 is a 2D HMQC spectrum of the correlations of the aliphaticprotons and carbons (Zoom-in of FIG. 17) in Peak A in d₆-DMSO;

FIG. 19 is a 2D HMQC spectrum of the correlations of the aromaticprotons and carbons in Peak A in d₆-DMSO;

FIG. 20 is a full range 2D HMBC spectrum of the correlations of theprotons and carbons in Peak A in d₆-DMSO;

FIG. 21 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and carbons in Peak A in d₆-DMSO;

FIG. 22 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and aromatic carbons in Peak A in d₆-DMSO;

FIG. 23 is a 2D HMBC spectrum of the correlations of the aromaticprotons and carbons in Peak A in d₆-DMSO;

FIG. 24 is a 2D HMBC spectrum of the correlations of the aromaticprotons and aliphatic carbons in Peak A in d₆-DMSO;

FIG. 25 is a HPLC Chromatogram using chromatographic method 4 showingre-injection of Peak B to determine its approximate purity;

FIG. 26 is a low resolution negative ion FAB-MS spectrum of Peak B;

FIG. 27 is a full range 400 MHz ¹H-NMR spectrum of Peak B in d₆-DMSO;

FIG. 28 is a 400 MHz ¹H-NMR spectrum of the aliphatic region of Peak Bin d₆-DMSO;

FIG. 29 is a 400 MHz ¹H-NMR spectrum of the aromatic region of Peak B ind₆-DMSO;

FIG. 30 is a full range 400 MHz 2D COSY ¹H-NMR spectrum of Peak B ind₆-DMSO;

FIG. 31 is a 400 MHz 2D COSY ¹H-NMR spectrum of the aliphatic region ofPeak B in d₆-DMSO;

FIG. 32 is a 400 MHz 2D COSY ¹H-NMR spectrum of the aromatic region ofPeak B in d₆-DMSO;

FIG. 33 is a full range 100 MHz ¹³C-NMR spectrum of Peak B in d₆-DMSO;

FIG. 34 is a 100 MHz ¹³C-NMR spectrum of the aliphatic region (0-38 ppm)of Peak B in d₆-DMSO;

FIG. 35 is a 100 MHz ¹³C-NMR spectrum of the aliphatic region (43-50ppm) of Peak B in d₆-DMSO;

FIG. 36 is a 100 MHz ¹³C-NMR spectrum of the aromatic region of Peak Bin d₆-DMSO;

FIG. 37 is a 100 MHz ¹³C-NMR spectrum of the carbonyl region of Peak Bin d₆-DMSO;

FIG. 38 is a full range 2D HMQC spectrum of the correlations of theprotons and carbons of Peak B in d₆-DMSO;

FIG. 39 is a 2D HMQC spectrum of the correlations of the aliphaticprotons and carbons in Peak B in d₆-DMSO;

FIG. 40 is a 2D HMQC spectrum of the correlations of the aliphaticprotons and carbons (Zoom in of FIG. 39) in Peak B in d₆-DMSO;

FIG. 41 is a 2D HMQC spectrum of the correlations of the aromaticprotons and carbons in Peak B in d₆-DMSO;

FIG. 42 is a full range 2D HMBC spectrum of the correlations of theprotons and carbons in Peak B in d₆-DMSO;

FIG. 43 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and carbons in Peak B in d₆-DMSO;

FIG. 44 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and aromatic carbons in Peak B in d₆-DMSO;

FIG. 45 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and carbonyl carbons in Peak B in d₆-DMSO;

FIG. 46 is a 2D HMBC spectrum of the correlations of the aromaticprotons and carbons in Peak B in d₆-DMSO; and

FIG. 47 is a 2D HMBC spectrum of the correlations of the aromaticprotons and aliphatic carbons in Peak A in d₆-DMSO.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention will now be described with reference to the embodimentsset forth herein. These embodiments are intended to illustrate theinvention and are not meant to limit the scope of the invention, whichis defined by the claims.

In one aspect of the present invention, a compound represented byFormula I is provided.

wherein the bond represented by the wavy line may be a single or doublebond such that when the wavy line is a single bond, R₁ may be selectedfrom the group consisting of hydrogen, sulfate and glucoronide or otheresters, and when the wavy line is a double bond, R₁ does not exist; R₂is lower alkyl; R₃ may be selected from the group consisting ofhydrogen, sulfate and glucuronide or other esters; and R₄ through R₁₃may independently be selected from the group consisting of hydrogen,hydroxy, ketone, lower alkyl (C₁ to C₄), lower alkoxy (C₁ to C₄),halogen, and carbonyl groups. When R₁ is hydroxy, the hydroxy or estersubstituent may have either an α or a β orientation, with the βorientation being preferred. R₂ is preferably C₁ to C₄ alkyl, and morepreferably is methyl. R₄ through R₁₂ are preferably hydrogen. R₁₃ ispreferably hydrogen or ethynyl. R₁₄ may be selected from the groupconsisting of hydrogen, sulfate and glucoronide and other esters

The compound represented by Formula I is present in chemically pureform, (i.e., greater than about 90% pure). The compound represented byFormula I is preferably greater than about 95% pure, and is mostpreferably greater than about 99% pure.

A preferred compound is illustrated in Formula II:

Another preferred compound is illustrated in Formula III:

Compounds of the present invention may be present in a conjugated form.The conjugates may be various conjugates understood by those skilled inthe art, including, but not limited to, glucuronide and sulfate. Themost preferred conjugate is sulfate.

Compounds of the present invention may also be present as variouspharmaceutically acceptable salts including salts of the conjugatedcompound. The salts may be various salts understood by those skilled inthe art, including, but not limited to, sodium salts, calcium salts,magnesium salts, lithium salts, and amine salts such as piperazinesalts. The most preferred salts are sodium salts.

In another aspect, the present invention provides a composition ofmatter. The composition of matter comprises one or more compoundsaccording to the present invention.

In one embodiment, the composition of the invention includes at leastone additional pharmaceutically active ingredient. Examples ofadditional active ingredients include, but are not limited to, otherestrogenic compounds, androgenic compounds, progestin compounds,vasodilation agents, calcium salts, and vitamin D and its derivatives(e.g., calcitriol and mixtures and blends thereof) and mixtures andblends of the various compounds.

Examples of estrogenic compounds and compositions are set forth in U.S.patent application Ser. No. 09/524,132 filed on Mar. 10, 2000, which iscommonly assigned to the assignee of the present invention, thedisclosure of which is incorporated by reference herein in its entirety.Suitable estrogenic compounds include estrone, 17α-estradiol,17β-estradiol, equilin, 17α-dihydroequilin, 17β-dihydroequilin,equilenin, 17α-dihydroequilenin, 17β-dihydroequilenin,Δ^(8,9)-dehydroestrone, 17α-Δ^(8,9)-dehydroestradiol,17β-Δ^(8,9)-dehydroestradiol, ethinyl estradiol, estradiol valerate,6-OH equilenin, 6-OH 17α-dihydroequilenin, 6-OH 17β-dihydroequilenin,and mixtures, conjugates and salts thereof, and the estrogen ketones andtheir corresponding 17α- and 17β-hydroxy derivatives. The estrogeniccompounds may also be present as conjugated estrogens. The conjugatesmay be various conjugates understood by those skilled in the art,including, but not limited to, sulfate and glucuronide. The mostpreferred estrogen conjugates are estrogen sulfates. The estrogeniccompounds may also be present estrogen conjugates. In one embodiment,the estrogenic compounds are present as salts of estrogen conjugates.The salts may be various salts understood by those skilled in the art,including, but not limited to, sodium salts, calcium salts, magnesiumsalts, lithium salts, and piperazine salts. The most preferred salts aresodium salts. The estrogenic compounds can be derived from natural andsynthetic sources.

Suitable androgenic compounds include methyltestosterone, androsterone,androsterone acetate, androsterone propionate, androsterone benzoate,androsteronediol, androsteronediol-3-acetate,androsteronediol-17-acetate, androsteronediol-3-17-diacetate,androsteronediol-17-benzoate, androsteronediol-3-acetate-17-benzoate,androsteronedione, dehydroepiandrosterone, sodium dehydroepiandrosteronesulfate, dromostanolone, dromostanolone propionate, ethylestrenol,fluoxymesterone, methyl testosterone, nandrolone phenpropionate,nandrolone decanoate, nandrolone furylpropionate, nandrolonecyclohexane-propionate, nandrolone benzoate, nandrolonecyclohexanecarboxylate, oxandrolone, oxymetholone, stanozolol,testosterone, testosterone decanoate, 4-dihydrotestosterone,5α-dihydrotestosterone, testolactone, 17α-methyl-19-nortestosterone andpharmaceutically acceptable esters and salts thereof, and combinationsof any of the foregoing.

Suitable vasodilation compounds include alpha andrenergic antagonists.Exemplary α-adrenergic compounds include phentolamine,phenoxybenzalamine, tolazoline, doxazosin, dibenamine, prazosin,prazosin hydrochloride, phenoxybenzamine and the like. Preferably,phentolamine is used and can form pharmaceutically acceptable salts withorganic and inorganic acids, as described, for example, in U.S. Pat. No.6,001,845 to Estok, the disclosure of which is incorporated herein byreference in its entirety. Preferably phentolamine mesylate orphentolamine hydrochloride is used. Other vasodilation compounds includephosphodiesterase type 5 inhibitors (e.g., sildenafil), prostaglandin Bcompounds (e.g., alprostadil), thymoxamine, bromocriptine, yohimbine,paperverine, apomorphine, organic nitrates, imipramine, verapamil,naftidrofuryl, and isoxsuprine. Combinations of the various vasodilationcompounds may be used.

Examples of progestins are set forth in U.S. Pat. No. Re. 36,247 toPlunkett et al., the disclosure of which is incorporated herein byreference in its entirety. Suitable progestin compounds includedesogestrel, dydrogesterone, ethynodiol diacetate, medroxyprogesterone,levonorgestrel, medroxyprogesterone acetate, hydroxyprogesteronecaproate, norethindrone, norethindrone acetate, norethynodrel,allylestrenol, 19-nortestosterone, lynoestrenol, quingestanol acetate,medrogestone, norgestrienone, dimethisterone, ethisterone, cyproteroneacetate, chlormadinone acetate, megestrol acetate, norgestimate,norgestrel, desogestrel, trimegestone, gestodene, nomegestrol acetate,progesterone, 5α-pregnan-3β,20α-diol sulfate, 5α-pregnan-3β,20β-diolsulfate, 5α-pregnan-3β-ol-20-one, 16,5α-pregnen-3β-ol-20-one,4-pregnen-20β-ol-3-one-20-sulfate and mixtures thereof.

Calcium salts may include, without limitation, organic acid salts ofcalcium such as calcium citrate, calcium lactate, calcium fumurate,calcium acetate, and calcium glycerophosphate, as well as inorganicsalts such as calcium chloride, calcium phosphate, calcium sulphate, andcalcium nitrate.

Pharmaceutically acceptable salts, solvates, hydrates, and polymorphsmay be formed of any of the active ingredients employed in thecomposition of the invention. The invention also encompasses embodimentsin which the composition of matter defined herein is included in variousquantities in combination with known pharmaceutically acceptedformulations. For example, the composition of matter of the inventionmay be incorporated into various known estrogen-containing drug productssuch as PREMARIN® (conjugated estrogens tablets USP made commerciallyavailable by Wyeth-Ayerst Laboratories of Philadelphia, Pa. Thecomposition of matter of the invention may also be employed as part of acontinuous estrogen-progestin therapy regimen such as that described byU.S. Pat. No. Re. 36,247 to Plunkett et al. and made commerciallyavailable as PREMPRO® (conjugated estrogens/medroxyprogesterone acetate)and PREMPHASE® (conjugated estrogens/medroxyprogesterone acetate) byWyeth-Ayerst Laboratories.

The present invention also encompasses pharmaceutically acceptable drugproducts comprising a composition of matter of the present invention andat least one pharmaceutically acceptable carrier, diluent, or excipient,the selection of which are known to the skilled artisan. The drugproduct formulations can be in various forms such as, for example,tablets; effervescent tablets; pills; powders; elixirs; suspensions;emulsions; solutions; syrups; soft and hard gelatin capsules;transdermal patches; topical gels, creams and the like; suppositories;sterile injectable solutions; and sterile packaged powders, sublingualtablets, buccal tablets, and buccal adhesive systems.

In certain embodiments, the drug product is present in a solidpharmaceutical composition that may be suitable for oral administration.A solid composition of matter according to the present invention may beformed and may be mixed with an excipient, diluted by an excipient orenclosed within such a carrier which can be in the form of a capsule,sachet, tablet, paper, or other container. When the excipient serves asa diluent, it may be a solid, semi-solid, or liquid material which actsas a vehicle, carrier, or medium for the composition of matter.

Various suitable excipients will be understood by those skilled in theart and may be found in the National Formulary 19, pages 2404-2406(2000), the disclosure of pages 2404 to 2406 being incorporated hereinin their entirety. For example, the drug product formulations mayinclude lubricating agents such as, for example, talc, magnesiumstearate and mineral oil; wetting agents; emulsifying and suspendingagents; binding agents such as starches, gum arabic, microcrystallinecellulose, cellulose, methylcellulose, and syrup; anticaking agents suchas calcium silicate; coating agents such as methacrylates and shellac;preserving agents such as methyl- and propyl hydroxybenzoates;sweetening agents; or flavoring agents. Polyols, buffers, and inertfillers may also be used. Examples of polyols include, but are notlimited to, mannitol, sorbitol, xylitol, sucrose, maltose, glucose,lactose, dextrose, and the like. Suitable buffers encompass, but are notlimited to, phosphate, citrate, tartarate, succinate, and the like.Other inert fillers which may be used encompass those which are known inthe art and are useful in the manufacture of various dosage forms. Ifdesired, the solid formulations may include other components such asbulking agents and/or granulating agents, and the like. The drugproducts of the invention may be formulated so as to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well known in theart.

To form tablets for oral administration, the composition of matter ofthe present invention may be made by a direct compression process. Inthis process, the active drug ingredients may be mixed with a solid,pulverant carrier such as, for example, lactose, saccharose, sorbitol,mannitol, starch, amylopectin, cellulose derivatives or gelatin, andmixtures thereof, as well as with an antifriction agent such as, forexample, magnesium stearate, calcium stearate, and polyethylene glycolwaxes. The mixture may then be pressed into tablets using a machine withthe appropriate punches and dies to obtain the desired tablet size. Theoperating parameters of the machine may be selected by the skilledartisan. Alternatively, tablets for oral administration may be formed bya wet granulation process. Active drug ingredients may be mixed withexcipients and/or diluents. The solid substances may be ground or sievedto a desired particle size. A binding agent may be added to the drug.The binding agent may be suspended and homogenized in a suitablesolvent. The active ingredient and auxiliary agents may also be mixedwith the binding agent solution. The resulting dry mixture is moistenedwith the solution uniformly. The moistening typically causes theparticles to aggregate slightly, and the resulting mass is pressedthrough a stainless steel sieve having a desired size. The mixture isthen dried in controlled drying units for the determined length of timenecessary to achieve a desired particle size and consistency. Thegranules of the dried mixture are sieved to remove any powder. To thismixture, disintegrating, antifriction, and/or anti-adhesive agents areadded. Finally, the mixture is pressed into tablets using a machine withthe appropriate punches and dies to obtain the desired tablet size. Theoperating parameters of the machine may be selected by the skilledartisan.

If coated tablets are desired, the above-prepared cores may be coatedwith a concentrated solution of sugar or cellulosic polymers, which maycontain gum arabic, gelatin, talc, titanium dioxide, or with a lacquerdissolved in a volatile organic solvent, aqueous solvent, or a mixtureof solvents. To this coating, various dyes may be added in order todistinguish among tablets with different active compounds or withdifferent amounts of the active compound present. In a particularembodiment, the active ingredient may be present in a core surrounded byone or more layers including enteric coating layers.

Soft gelatin capsules may be prepared in which capsules contain amixture of the active ingredient and vegetable oil. Hard gelatincapsules may contain granules of the active ingredient in combinationwith a solid, pulverulent carrier, such as, for example, lactose,saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin,cellulose derivatives, and/or gelatin.

In one preferred embodiment, the formulation is in the form oforally-administered tablets which contain the composition of matter ofthe present invention as set forth herein along with the followinginactive ingredients: calcium phosphate tribasic, calcium sulfate,carnauba wax, cellulose, glyceryl monooleate, lactose, magnesiumstearate, methylcellulose, pharmaceutical glaze, polyethylene glycol,stearic acid, sucrose, and titanium dioxide. Such ingredients may bepresent in amounts similar to those present PREMARIN® (conjugatedestrogens tablets, USP) made commercially available by Wyeth-AyerstLaboratories of Philadelphia, Pa. Tablets employing the activeingredients of the invention may contain excipients similar to thosecontained in the 0.3 mg., 0.625 mg., and 1.25 mg tablets of PREMARIN®(conjugated estrogens tablets, USP).

Liquid preparations for oral administration may be prepared in the formof syrups or suspensions, e.g., solutions containing an activeingredient, sugar, and a mixture of ethanol, water, glycerol, andpropylene glycol. If desired, such liquid preparations may containcoloring agents, flavoring agents, and saccharin. Thickening agents suchas carboxymethylcellulose may also be used.

In the event that the above formulations are to be used for parenteraladministration, such a formulation may comprise sterile aqueousinjection solutions, non-aqueous injection solutions, or both comprisingthe composition of matter of the present invention. When aqueousinjection solutions are prepared, the composition of matter may bepresent as a water-soluble pharmaceutically acceptable salt. Parenteralpreparations may contain anti-oxidants, buffers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient. Aqueous and non-aqueous sterile suspensions mayinclude suspending agents and thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example sealedampules and vials. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules and tablets of the kindpreviously described.

In a preferred embodiment, the drug product of the present invention isin the form of an injectable solution containing a predetermined amount(e.g., 25 mg) of the composition of matter in a sterile lyphilized cakewhich also contains lactose, sodium citrate, and simethicone. The pH ofa solution containing the above ingredients may be adjusted using asuitable buffer (e.g., sodium hydroxide or hydrochloric acid).Reconstitution may be carried out according to known methods, e.g.,using a sterile diluent (5 mL) containing 2 percent benzyl alcohol insterile water. A preferred injectable solution is similar to Premarin®Intravenous made commercially available by Wyeth-Ayerst Laboratories.

The composition of matter also may be formulated such that it issuitable for topical administration (e.g., vaginal cream). Theseformulations may contain various excipients known to those skilled inthe art. Suitable excipients may include, but are not limited to, cetylesters wax, cetyl alcohol, white wax, glyceryl monostearate, propyleneglycol monostearate, methyl stearate, benzyl alcohol, sodium laurylsulfate, glycerin, mineral oil, water, carbomer, ethyl alcohol, acrylateadhesives, polyisobutylene adhesives, and silicone adhesives.

In a preferred embodiment, the drug product is in the form of a vaginalcream containing the composition of matter as set forth herein presentin a nonliquefying base. The nonliquefying base may contain variousinactive ingredients such as, for example, cetyl esters wax, cetylalcohol, white wax, glyceryl monostearate, propylene glycolmonostearate, methyl stearate, benzyl alcohol, sodium lauryl sulfate,glycerin, and mineral oil. Such composition may be formulated similar toPremarin® Vaginal Cream made commercially available by Wyeth-AyerstLaboratories.

Dosage units for rectal administration may be prepared in the form ofsuppositories which may contain the composition of matter in a mixturewith a neutral fat base, or they may be prepared in the form ofgelatin-rectal capsules which contain the active substance in a mixturewith a vegetable oil or paraffin oil.

In another aspect, the present invention relates to methods of treatingmammals (e.g., man) in need of treatment. The methods includeadministering an effective amount of a composition of matter as definedherein to the mammal in need of treatment. The methods may be used for anumber of treatments such as, but not limited to, vasomotor symptoms;atrophic vaginitis; osteoporosis; hypoestrogenism due to hypogonadism,castration, or primary ovarian failure; breast cancer in selectedpersons with metastatic disease; advanced androgen-dependent carcinomaof the prostate; abnormal uterine bleeding; and kraurosis vulvae. Theadministration may be cyclic, occurring for one or more short periods oftime or courses of treatment (i.e. short-term use). Alternatively, theadministration may be continuous, occurring over extended periods oftime (i.e. long-term use). One example of long-term use would be fromthe onset of menopause until death. Cyclic and continuous administrationmay be either uninterrupted or interrupted. Uninterrupted administrationoccurs one or more times daily such that there is no break in treatment.Interrupted administration occurs other than daily, for example arepeated course of treatment including three weeks of daily treatmentfollowed by one week of no treatment.

EXAMPLES

The present invention will now be described in greater detail withrespect to the following numbered examples. In the examples, “mL” meansmilliliter, “° C.” means degrees Celcius, “mM” means millimoles/liter,“M” means moles/liter, “Å” means angstrom, “μm” means micrometer, “nm”means nanometer, “mm” means millimeter, “mg” means milligram, and “m/z”means mass to charge ratio. These examples are for illustrating theinvention and are not intended to limit the invention as set forth bythe claims.

A list of instruments and equipment employed in the examples are asfollows:

1. HPLC Chromatographic Procedures

-   -   a. Analytical scale chromatographic system        -   1. HP1100 Diode-array detector        -   2. HP1100 Quaternary HPLC pump        -   3. Shimadzu, Model RF-551, fluorescence detector        -   4. HP1100 Thermostatically controlled column compartment    -   b. Semi-prep scale chromatographic system        -   1. HP1100 HPLC chromatographic system        -   2. HP1100 Diode-array detector        -   3. HP1100 Quaternary HPLC pump        -   4. HP1100 Thermostatically controlled column compartment    -   c. Prep scale chromatographic system        -   1. Waters Delta Prep 4000 chromatographic system        -   2. Waters 2487 UV detector        -   3. Waters fraction collector II        -   4. Waters PrepLC 40 mm radial compression assembly        -   5. Waters Nova-Pak HR C₁₈ gun radial compression column            segments, (2) 40 mm×100 mm segments with a 40 mm×10 mm guard            segment

2. Fraction Collection, Purification, and Crystallization

-   -   a. ISCO Foxy Jr., Fraction Collector    -   b. Büchi, Model R-124 rotary evaporator    -   c. Sep-Pak, SPE cartridges, Varian Bond Elut C₁₈    -   d. Waters fraction collector II

3. Mass Spectral Analyses

-   -   a. Fast Atom Bombardment (FAB-MS)        -   1. Instrument: VG Analytical ZAB 2-SE        -   2. Sample input: Cesium ion gun        -   3. Data system: VG Analytical 11-250J with PDP 11/73        -   4. Solvent: Methanol        -   5. Matrix: Glycerol/Thioglycerol/Triethylamine    -   b. High Resolution Mass Spectrometer (HR-MS)        -   1. Instrument: VG Analytical ZAB 2-SE        -   2. Sample input: Cesium ion gun        -   3. Data System: VG Analytical 11-250J with PDP 11/73        -   4. Solvent: Methanol        -   5. Matrix: Peak A: PEG 300 & PEG 400            -   Peak B: PEG 300 & m-nitrobenzyl alcohol

B. Chemicals, Reagents, and Analytical Materials

-   -   1. Chemicals and Reagents        -   a. Acetonitrile (ACN), HPLC grade        -   b. Methanol (MeOH), HPLC grade        -   c. Milli-Q water        -   d. Triethylamine (TEA), HPLC grade        -   e. tert-Butyl ammonium hydroxide (TBAH), 0.4 M, reagent            grade        -   f. Potassium phosphate monobasic, AR grade        -   g. Nitrogen gas, zero grade        -   h. Phosphoric acid, 85%        -   i. Hydrochloric acid, concentrated        -   j. Sodium hydroxide

2. Analytical Samples

-   -   Conjugated estrogens tablets, 1.25 mg, FDL lot #00426-064

3. Analytical Standards

-   -   a. Conjugated Estrogens Reference Standard (ten component),    -   Organics/LaGrange, Inc. (OLG) lot #C02322    -   b. Equilin sulfate, sodium salt, OLG lot#RD1810    -   c. Equilin sulfate, sodium salt, Diosynth lot#00004429    -   d. 17α-Dihydroequilin sulfate, sodium salt, OLG lot# RD1812    -   e. 17α-Dihydroequilin sulfate, sodium salt, Diosynth lot#58    -   f. Δ^(8,9)-Dehydroestrone, Proquina lot#9371-1/95    -   g. Δ^(8,9)-Dehydroestrone, lot# HS 30/95, supplier unknown    -   h. Δ^(8,9)-Dehydroestrone, sulfate+estrone sulfate, sodium        salts, lot#17-44, supplier unknown

Examples 1-4 Isolation of Compounds A and B

The compounds of Peaks A and B are isolated from a 10-componentconjugated estrogen available from Endeavor Pharmaceuticals ofWilmington, N.C. as follows:

Example 1 HPLC Chromatographic Assay Method 1 Analytical Scale

A standard solution containing about 0.03 mg/mL of Conjugated EstrogensDrug Substance may be prepared. The drug substance may be provided inpowder form or a powder may be formed by grinding tablets. Anappropriate amount of drug substance is weighed to yield 200 mL ofsolution. The drug substance is placed in a 200 ml volumetric flask. A61 mL volume of organic diluent is added to the flask and the flask ismechanically shaken for 15 minutes. About 100 mL of aqueous diluent isthen added to the flask and the flask is once again mechanically shakenfor 15 minutes. The resulting solution is diluted to volume with aqueousdiluent and mixed well. A portion of the solution is filtered through a0.45 μm PTFE filter.

A 50 mM phosphate buffer solution may be prepared using potassiumphosphate.

An aqueous diluent solution containing phosphate buffer and 0.4 M TBAHwith a volumetric ratio of 277:0.9 may be prepared. The pH can beadjusted to 3.0±0.1 using phosphoric acid.

An organic diluent solution containing acetonitrile and methanol with avolumetric ratio of 26.5:4 may be prepared.

A mobile phase may be prepared by mixing organic diluent and aqueousdiluent to form a solution with a volumetric ratio of 30.5:69.5,organic:aqueous.

When 1.25 mg tablets are to be analyzed, five washed and ground tabletsare placed into a 200 mL volumetric flask. A 61 mL volume of organicdiluent is added to the flask and the flask is mechanically shaken for15 minutes. About 100 mL of aqueous diluent is then added to the flaskand the flask is once again mechanically shaken for 15 minutes. Theresulting solution is diluted to volume with aqueous diluent and mixed.A portion of the solution is filtered through a 0.45 μm PTFE filter.

In this chromatographic analysis, an HPLC system with a column heaterequipped with a 3 μm, 15.0 cm×4.6 mm C₁₈ column and suitable UV detectorfor detection at 220 nm and diode array was employed. The flow rate wasset for 1.5 mL/minute and the column temperature was set for 25° C.

An example of a chromatographic procedure is as follows: Equal volumesof the standard solution and the sample preparations were separatelyinjected into the chromatographic systems. Peaks A and B were integratedand evaluated based on the peak area response for the chromatogram (areapercent).

Example 2 HPLC Chromatographic Separation Method 2 Semi-Prep Scale

A Mobile Phase A (aqueous) containing 15 mM TEA in 0.125% concentratedHCl may be prepared by combining 42 mL of TEA with 20 L of water andmixing well. To the resulting solution, 25 mL of concentrated HCl isadded and the pH is adjusted to approximately 7.0 with 1 N HCl or 1 NNaOH.

A Mobile Phase B (organic) containing 15 mM TEA in 0.125% concentratedHCl in acetonitrile may be prepared by combining 16.8 mL of TEA with 8 Lof acetonitrile and adding 10 mL of concentrated HCl. The resultingsolution is then mixed well.

A sample for Peak A collection containing a 30 mg/mL solution of17α-dihydroequilin sulfate, sodium salt in mobile phase A may beprepared.

In this chromatographic separation for Peak A collection, a semi-prepscale HPLC system equipped with an appropriate fraction collector, aWaters Symmetry C₁₈ (7.8 mm×300.0 mm), 7 μM column, and a suitable UVdetector for detection at 220 nm may be employed. The flow rate may beset for 5 mL/minute, the column temperature may be set to 40° C., andthe gradient elution profile may be as follows:

Time % Mobile Phase (Minutes) A B 0.0 83 17 42.0 83 17 42.1 50 50 47.050 50 47.1 83 17

An example of a chromatographic procedure is as follows: 150 μL portionsof the sample solution were separately injected into the chromatographuntil all of the sample solution had been injected. The fractioncontaining the peak at approximately 41 minutes was collected andlabeled as Peak A.

Example 3 HPLC Chromatographic Separation Method 3 Prep Scale

A Mobile Phase A (aqueous) containing 60 mM TEA in 0.5% concentrated HClmay be prepared by combining 168 mL of TEA with 20 L of water and mixingwell. To the resulting solution, 100 mL of concentrated HCl is added andthe pH is adjusted to approximately 3.0 with 1 N HCl or 1 N NaOH.

A Mobile Phase B (organic) containing 60 mM TEA in 0.5% concentrated HClin acetonitrile may be prepared by combining 84 mL of TEA with 10 L ofacetonitrile and adding 50 mL of concentrated HCl. The resultingsolution is then mixed well.

A sample solution for Peak A collection containing a 20 mg/mL solutionof 17α-dihydroequilin sulfate, sodium salt in mobile phase A may beprepared.

A sample solution for Peak B collection containing a 20 mg/mL solutionof equilin sulfate, sodium salt in mobile phase A may be prepared.

In this chromatographic separation for Peak A collection, a prep scaleHPLC system equipped with an appropriate fraction collector, two 40mm×100.0 mm radial compression C₁₈ column segments, a 40 mm×10 mm guardsegment and a suitable UV detector for detection at 220 nm with the fullscale absorbance set at 4.0 may be employed. The flow rate may be setfor 50 mL/minute, the temperature is preferably ambient temperature, andthe gradient elution profile may be as follows:

Time % Mobile Phase (Minutes) A B 0.0 83 17 43.0 83 17 43.1 40 60 52.040 60 52.1 83 17

In this chromatographic separation for Peak B collection, a prep scaleHPLC system equipped with an appropriate fraction collector, two 40mm×100.0 mm radial compression C18 column segments, a 40 mm×10 mm guardsegment and a suitable UV detector for detection at 220 nm with the fullscale absorbance set at 4.0 may be employed. The flow rate may be setfor 50 mL/minute, the temperature is preferably ambient temperature, andthe gradient elution profile may be as follows:

Time % Mobile Phase (Minutes) A B 0.0 80 20 35.0 80 20 35.1 40 60 41.040 60 41.1 80 20

An example of a chromatographic procedure for Peak A collection is asfollows: 10 mL portions of the sample solution for Peak A wereseparately injected into the chromatograph until all of the samplesolution had been injected. The fraction containing the peak atapproximately 39 minutes was collected and labeled as Peak A.

An example of a chromatographic procedure for Peak B collection is asfollows: 10 mL portions of the sample solution for Peak B wereseparately injected into the chromatograph until all of the samplesolution had been injected. The fraction containing the peak atapproximately 29 minutes was collected and labeled as Peak B.

Example 4 HPLC Chromatographic Assay Method 4 Analytical Scale

A Mobile Phase A (aqueous) containing 15 mM TEA in 0.125% concentratedHCl may be prepared by combining 4.2 mL of TEA with 2 L of water andmixing well. To the resulting solution, 2.5 mL of concentrated HCl isadded and the pH is adjusted to approximately 3.0 with 1 N HCl or 1 NNaOH.

A Mobile Phase B (organic) containing 15 mM TEA in 0.125% concentratedHCl in acetonitrile may be prepared by combining 4.2 mL of TEA with 2 Lof acetonitrile and adding 2.5 mL of concentrated HCl. The resultingsolution is then mixed well.

A mobile phase may be prepared using a gradient system or by manuallypreparing a mixture of 80% (v/v) Mobile Phase A and 20% (v/v) MobilePhase B.

A sample solution containing approximately 0.06 mg/mL conjugatedestrogens in mobile phase may be prepared by transferring one 1.25 mgtablet into a 25 mL volumetric flask. A 6 mL volume of Mobile Phase B isadded to the flask and the flask is mechanically shaken for 10 minutes.The resulting solution is diluted to volume with Mobile Phase A and thenmixed well. A portion of the solution is filtered through a 0.45 μm PTFEfilter.

In this chromatographic analysis, an HP1100 HPLC system with a columnheater equipped with a 3 μm, 15.0 cm×4.6 mm C₁₈ column and suitable UVdetector for detection at 220 nm and diode array was employed. The flowrate may be set for 1.5 mL/minute and the column temperature may be setfor 25° C.

An example of a chromatographic procedure is as follows: Equal volumesof the standard solution and the sample preparations were separatelyinjected into the chromatographic systems. Peaks A and B were integratedand evaluated based on the peak area response for the chromatogram (areapercent).

Examples 5 and 6 Characterization of Peaks A and B

Examples 5 and 6 detail the characterization of Peaks A and B found inthe Endeavor Pharmaceuticals 10-Component Conjugated Estrogens drugproduct (FIG. 1). These compounds were also found to be present incertain estrogen standards which were evaluated. Peak A was found to bepresent in the 17α-dihydroequilin sulfate, sodium salt standard (FIG. 2)and Peak B was found to be present in the equilin sulfate, sodium saltstandards (FIG. 3). Due to the simplicity of the standard materials, theindividual peaks were collected by chromatographic fraction collectionfrom the corresponding standard and upon purification, were isolated asyellowish amorphous materials.

The fractioned samples were analyzed using techniques such as massspectrometry (MS), and one-dimensional (1D) and two-dimensional (2D)nuclear magnetic resonance (NMR) spectrometry. Mass spectrometryutilized both low and high resolution fast atom bombardment massspectrometry (FAB-MS) to determine the accurate molecular weight andempirical formula of the compounds. For the NMR analyses, samples weredissolved in deuterated dimethyl sulfoxide (d₆-DMSO), which permittedthe hydroxyl protons to be visible during analysis. NMR techniquesincluded ¹H-NMR (proton), ¹³C-NMR (carbon), homonuclear correlationspectroscopy (COSY), distortionless enhancement by polarization transfer(DEPT), heteronuclear multiple quantum coherence (HMQC), andheteronuclear multiple bond correlation (HMBC). 2D COSY analyses helpeddetermine the correlation of neighboring protons to one another, whileDEPT analyses determined the assignment of carbon type. 2D HMQC analysesprovided information about what protons are attached to what carbons,and 2D HMBC analyses showed longer range coupling of the proton andcarbon atoms (usually 2 to 4 bonds away).

Example 5 Peak A Characterization

Separation and Isolation

Peak A was isolated as a triethyl ammonium salt due to the ion-pairingagent of the mobile phase of the HPLC chromatographic method describedabove in Example 2. After the fraction was collected, most of the ACNwas removed by rotary evaporation, and the fraction was furtherconcentrated using a C₁₈ SPE cartridge, washed with water, and elutedwith approximately 10 mL of methanol. The fraction was then brought todryness under a stream of dry nitrogen. Using the HPLC method describedin above Example 4, a small portion of the 5.3 mg of Peak A isolated fortesting by MS and NMR was redissolved in mobile phase and injected intothe HPLC system to determine the purity of the fraction. This injectionof Peak A showed a purity of about 89% (FIG. 4).

Mass Spectral Analyses

Preliminary negative ion FAB-MS spectral data of the isolated fractionof Peak A indicated that the molecular weight was approximately 363 m/z(FIG. 5). A negative ion HR-MS study indicated a mass of 363.0909 amuthat compares well with the calculated mass of 363.0902 amu for theproposed molecular formula of C₁₈H₁₉O₆S₁ for Peak A.

Proton (¹H) and 2D COSY Nuclear Magnetic Resonance Spectroscopy

The ¹H-NMR and the 2D COSY spectra of Peak A in deuterated dimethylsulfoxide (d₆-DMSO) are shown in FIGS. 6-8 and 9-11, respectively. Thepeak assignments, based upon the proton NMR spectra and COSY spectralcouplings, are shown in Table 1 and are consistent with the proposedstructure of Peak A.

Carbon (¹³C), 2D HMQC, and 2D HMBC Nuclear Magnetic Resonance

The ¹³C-NMR, HMQC, and HMBC spectra of Peak A in deuterated dimethylsulfoxide (d₆-DMSO) are shown in FIGS. 12-15, 16-19, and 20-24,respectively. In order to collect the data more quickly and with agreater signal to noise ratio, the ¹³C-NMR spectrum was obtainednon-quantitatively, and integrations were not performed. Peakassignments based upon the carbon NMR, HMQC, and HMBC spectralinterpretations are shown in Table 2 and were consistent with theproposed structure of Peak A. 2D HMBC data can be more difficult tointerpret, since it is possible that all crosspeaks are not observed.HMBC signals may typically occur with H—C connectivities that are 2 to 4bonds removed, but also can detect some 1 to 2 bond connections.

TABLE 1 Summary Table of Proton NMR and COSY Band Assignments ChemicalShift Tenative Multi- Number Of COSY (ppm) plicty* Protons Couplings**Assignment 0.52 s 3  12b 18 1.17 t 9 19 20 1.52 m 1 14, 15b, 16a, 16b 15a 1.60 m 1 15a, 15b, 16b  6a 1.73 m 1 11a, 11b, 12b 12a 2.04 m 1 11a,11b, 12a, 18 12b 2.16 m 1 14, 15a, 16a 15b 2.27 m 1 15a, 16a, 17 16b2.50 — — — solvent-DMSO 2.99 m 1 12a, 12b, 11b 11a 3.09 q 6 20 19  3.10d 1 15a, 15b 14  3.13 m 1 12a, 12b 11b 3.17 — — — solvent-MeOH 3.33 — —— solvent-H₂O 3.76 t 1 16b, 17-OH 17  4.09 — — — solvent-MeOH 4.50 d(w)1 17 17(OH) 6.62 s 1 — 7 7.30 d of d 1 1, 4 2 7.75 d 1  2 1 7.85 d(w) 1 2 4 8.90 bs 1 — NH⁺ 9.64 s 1 —  6(OH) *s—singlet, d—doublet, t—triplet,q—quartet, m—multiplet, b—broad, w—weak **weaker couplings areunderlined

TABLE 2 Summary Table of Carbon NMR, HMQC, and HMBC Peak AssignmentsChemical Shift Tenative Number Of HMQC HMBC (ppm) Carbons CouplingsCouplings Assignments 8.6 3 1.17 — 20 15.8 1 0.52 12, 13, 17 18 23.4 12.99, 3.13 8, 9, 13 11 24.6 1 1.52, 2.16 8, 13, 16 15 29.2 1 1.73, 2.049, 11, 13, 18 12 32.9 1 1.60, 2.27 15, 17 16 39-40 — — — solvent-DMSO44.4 1 — — 13 44.5 1 3.10 8, 9, 12, 13, 15, 14 18 45.7 1 3.09 — 19 48.5— — — solvent-MeOH 77.3 1 3.76 13, 15, 18 17 107.5 1 6.62 5, 6, 9, 14 7111.7 1 7.85 2, 3, 6, 10 4 119.9 1 — — 9 121.5 1 7.30 10 2 123.6 1 7.753, 5, 9, 10 1 123.7 1 — — 5 129.4 1 — — 10 136.0 1 — — 8 149.4 1 — — 3150.7 1 — 6 — — 4.50 13, 16, 17 17(OH) — — 9.64 5, 6, 7  6(OH)1D and 2D NMR Spectral Interpretation

Peak A is a derivative of dihydroequilenin, which contains five aromaticprotons and ten aliphatic protons. The ¹H-NMR spectrum exhibits the tenexpected aliphatic protons, but only four main signals were observed inthe aromatic region (6.5-8.0 ppm) of the ¹H-NMR (FIG. 8) thatcorresponded to a 1:1:1:1 ratio. Based upon the splitting expected fromthe proposed structure for Peak A, these signals were consistent with adihydroequilenin based ring structure substituted at one of the aromaticprotons. The four aromatic ¹H-NMR signals showed a strong singlet and astrong doublet, and a second singlet and doublet, which are weakly splitinto doublets.

Substitution at each of the possible aromatic positions may create adistinct splitting pattern. Substitution at the 1-position may create asingle pair of strong doublets (H6 and H7) with a strong COSYcorrelation and a pair of singlets (H4 and H2) which may exhibit a weakCOSY correlation and be weakly split by each other. Substitution at the2-position may create but a single pair of strong doublets (H6 and H7)with a strong COSY correlation and a pair of singlets (H4 and H1).Substitution of the aromatic ring system at the 4-position may create apattern of 2 strong pairs of doublets with strong COSY correlations inthe spectrum. Substitution at the 6- or 7-position may create but asingle pair of strong doublets (H1 and H2) with a strong COSYcorrelation and a pair of singlets (H4 and H6 or H7). The H4 proton maybe expected to interact weakly with the H2 proton exhibiting a weak COSYcorrelation and causing the H2 doublet and the H4 singlet to be weaklysplit by each other.

Based upon the splitting pattern of the aromatic protons, substitutionof the hydroxyl group in Peak A may be at either the H6 or H7 position.The pair of doublets at 7.75 and 7.30 ppm for H1 and H2, respectively,is shown to be adjacent from the 2D COSY spectrum (FIG. 11). H2 and H4at 7.85 ppm exhibited a weak COSY correlation that caused H2 to appearas a doublet of doublets due to splitting by both H1 and H4; and H4 as astrong singlet weakly split to a doublet. The assignments of thecorresponding carbons C1, C2, and C4 were based upon the HMQC spectra(FIG. 19) at 123.6, 121.5, and 111.7 ppm, respectively. The otheraromatic ring contains only one proton at 6.62 ppm for H6 or H7, whichwas observed as a singlet as expected since no other protons are nearbyto cause splitting. The corresponding carbon signal was assigned fromthe HMQC correlations at 107.5 ppm (FIG. 19).

In the ¹³C-NMR aromatic region (100-170 ppm) (FIG. 15), there were fourlarge signals and six smaller signals. Protonated carbons may typicallyhave larger signals than non-protonated carbons and that was used indifferentiating among the aromatic carbon atoms. This was verified byobservation of only four HMQC signals in this region, which occur onlyfor carbons with directly attached protons, in the aromatic region (FIG.19). The remaining six aromatic carbon signals did not have HMQC peaks,and are therefore, non-protonated.

Two of the six non-protonated signals are shifted downfield to about 150ppm (149.4 and 150.7 ppm), which may be typical of aromatic carbon atomsattached to an oxygen atom. This fits the proposed structure with thenormal 3-position hydroxy sulfate ester and the proposed hydroxylsubstitution on an aromatic position. The HMBC spectrum (FIG. 23) showscorrelations of the carbon signals at 149.4 ppm to H1 and H4 and thecarbon signal at 150.7 ppm to H4 and either H6 or H7. Based upon thosecorrelations the signal at 149.4 ppm must be C3. The signal at 150.7 ppmmust be C6 or the H4 correlation would have been a weak 4-bondcorrelation. Thus, the substitution is at the 6-position and thearomatic proton at 6.62 ppm and the aromatic carbon at 107.5 ppm areassigned H7 and C7, respectively. The remaining four non-protonatedcarbon atoms (119.9, 123.7, 129.4, and 136.0 ppm) match the number ofbridging non-protonated carbon atoms expected for the proposedstructure. Assignment of these four signals can be made from HMBCcorrelations (FIG. 23). H2 shows a single strong 3-bond correlation tothe carbon signal at 129.4 ppm and is assigned C10 being the onlybridging carbon atom within 3 bonds of H2. H1 exhibits three strong3-bond correlations at 119.9, 123.7, and 149.4 (C3) ppm and one weak2-bond correlation to 129.4 (C 10) ppm. H4 exhibits HMBC correlations to121.5 (C2), 129.4 (C10), 149.4 (C3), and 150.7 (C6) ppm. H7 exhibitsHMBC correlations to aromatic signals at 119.9, 123.7, and 150.7 (C6)ppm. Based upon these correlations, the carbon signals at 119.9 and123.7 ppm are for C5 and C9, but their exact assignments are not yetestablished.

There is a single strong signal downfield in the ¹H-NMR spectrum at 9.64ppm (FIG. 8). This region is typical of aromatic phenolic protons andthis signal is assigned as H6(OH). HMBC spectrum for this protonexhibits correlations at 107.5 (C7), 123.7, and 150.7 (C6) ppm (FIG.23). Based upon these correlations and the relationships of the otherprotons the signal at 123.7 ppm must be C5 and thus the signal at 119.9ppm must be C9. This leaves only the aromatic carbon signal at 136.0 ppmunassigned. Thus, the remaining aromatic signal by process ofelimination was assigned as C8.

The methyl region (0.5 to 1.5 ppm) of the ¹H-NMR spectrum (FIG. 7) showsa strong methyl signal split into a triplet at 1.17 ppm that isindicative of the methyl proton (H20) of the triethyl ammonium cation.This signal shows a strong COSY correlation to the quartet signal at3.09 ppm for the protons (H19) of the methylene group (FIG. 10). TheHMQC correlation spectrum (FIG. 17) showed corresponding carbon atoms at8.6 and 45.7 ppm for C20 and C19, respectively. The amine proton (NH⁺)of the cation was expected to have a ¹H-NMR chemical shift of about 8.0to 9.5 ppm; however, amines have the problem of slow exchange and oftenare not seen, or are only seen as a small broad peak in this region. TheNH proton in the ¹H-NMR spectrum was observed as a single broad signalat about 8.90 ppm for this compound (FIG. 8).

In the ¹³C-NMR aliphatic region (0-100 ppm) (FIGS. 13-14), there weretwo strong signals at 8.6 and 45.7 ppm for the triethyl ammonium cation,and eight signals for the aliphatic carbons of Peak A. The proposedstructure for Peak A contains eight aliphatic carbon atoms. The eightaliphatic carbon signals were observed at 15.8, 23.4, 24.6, 29.2, 32.9,44.4, 44.5, and 77.3 ppm. Seven of the eight signals show HMQCcorrelations to proton signals (FIGS. 17-18). Only the carbon signal at44.4 ppm did not exhibit a correlation to any proton signal and wasconsidered a bridging carbon. The proposed structure has one bridgingaliphatic carbon atom, and thus the peak at 44.4 ppm was assigned asC13. The three carbon signals at 15.8, 44.5, and 77.3 ppm eachcorrelated to a single proton signal whereas the other four carbonsignals observed at 23.4, 24.6, 29.2, and 32.9 ppm each correlated totwo proton signals. This may be the case in saturated aliphatic ringsystems since the two protons of the methylene groups are present indiffering electronic environments and thus, exhibit different chemicalshifts.

Inspection of the three carbon signals with a single proton HMQCcorrelation shows that the signal at 15.8 ppm is in the expected methylregion for ¹³C-NMR and correlates by HMQC to the proton signal at 0.52ppm. These protons exhibited the expected integration ratio for a methylgroup of 3:1 relative to the individual aromatic protons and areassigned as H18 based on the chemical shift and the HMQC correlationsand is the only methyl group in the proposed structure for Peak A. Thesignal for the carbon atom (C17) attached to the hydroxyl group isexpected to shift downfield relative to the other aliphatic signals, asdescribed for the aromatic carbon signals. Thus, the carbon signalobserved at 77.3 ppm was assigned as C17 based upon chemical shift andthe HMQC correlated proton signal at 3.76 ppm was assigned as H17. Thereis only one remaining carbon atom with one proton, thus, the signal at44.5 ppm was assigned as C14 and the proton signal at 3.10 ppmcorrelated to it by HMQC was H14.

The remaining four aliphatic carbon signals each exhibited two HMQCcorrelations to proton signals. The carbon signal at 23.4 ppm correlatesto the protons at 2.99 and 3.13 ppm. The COSY spectrum (FIG. 10) ofthese two protons show that they couple to the proton signals at 1.73and 2.04 ppm indicating the two sets are adjacent. The HMQC spectrum(FIG. 17) shows these protons both correlate to the carbon signal at29.2 ppm. The carbon signal at 24.6 ppm correlates to the protons at1.52 and 2.16 ppm. The COSY spectrum of these two protons show that theycouple to the proton signals at 1.60 and 2.27 ppm, indicating the twosets are adjacent. The HMQC spectrum shows these protons both correlateto the carbon signal at 32.9 ppm. These observations are consistent withthe proposed structure of Peak A that has two sets of adjacent methylenegroups at C11 and C12, and at C15 and C16.

HMBC couplings (FIGS. 21-22) can be used to ascertain the identity andposition of each of the four methylene groups. The bridging carbon at119.9 (C9) ppm correlates to only the protons of, the carbon signals at23.4 and 29.2 ppm (FIG. 22). This observation verifies that the carbonsignals at 23.4 and 29.2 ppm must be assigned as C11 and C12 and thatthe other two carbon signals at 24.6 and 32.9 ppm must be assigned asC15 and C16, but their exact assignments have not yet been established.The proton at 0.52 (H18) ppm shows a HMBC correlation to the carbonsignals at 29.2, 44.4 (C13), and 77.3 (C17) ppm (FIG. 21). Based uponthis, the carbon signal at 29.2 ppm was assigned as C12 and thus, thecarbon signal at 23.4 ppm must be C11. Based upon the HMQC correlations(FIG. 18) of each of these carbon signals, the proton signals at 2.99and 3.13 ppm can be assigned as H11a and H12b, respectively, and theprotons at 1.73 and 2.04 ppm can be assigned as H12a and H12b,respectively. The proton at 3.76 (C17) ppm shows COSY correlations tothe protons at 2.27 ppm and 4.50 ppm (FIG. 10). Based upon the COSYcorrelations of the proton signal at 2.27, it must be adjacent to H17and thus the carbon signal at 32.9 ppm must be C16 and the carbon signalat 24.6 ppm must be C15. Based upon the HMQC correlations (FIG. 18) ofeach of the carbon signals, the proton signals at 1.52 and 2.16 ppm canbe assigned as H15a and H15b, respectively, and the protons at 1.60 and2.27 ppm can be assigned as H16a and H16b, respectively. The proton at4.50 ppm adjacent to the 3.76 (C17) ppm proton must be H17(OH), whichwould be expect to shift downfield being directly attached to an oxygenatom. HMBC correlations of the proton at 4.50 ppm (H17(OH)) exhibitcorrelations to 32.9 (C16), 44.4 (C13), and 77.3 (C17) ppm carbonsignals (FIG. 21). Other HMBC correlations are detailed in Table 2 aboveand are consistent with the proposed structure of Peak A.

Additional signals in the proton NMR spectrum (FIGS. 7-8) were observedfrom water at 3.33 ppm, DMSO at 2.50 ppm, a small amount of methanol at3.17 and 4.09 ppm, and other assorted small, unidentified aromatic andaliphatic “impurity” signals.

In addition to the expected aromatic and aliphatic signals of Peak A,there are two other significant HMQC signals (FIG. 17). The solvent(d₆-DMSO) was observed at 39.5 ppm (HMQC peak at 2.50 ppm), and themethanol was observed at 48.5 ppm (HMQC peak at 3.17 ppm).

Based on the observed NMR data the structure of Peak A corresponds wellto the proposed structure.

Example 6 Peak B Characterization

Separation and Isolation

Peak B was isolated as a triethyl ammonium salt due to the ion-pairingagent of the mobile phase of the HPLC chromatographic method describedabove in Example 3. After the fraction was collected, most of the ACNwas removed by rotary evaporation, and the fraction was furtherconcentrated using a C₁₈ SPE cartridge, washed with water, and elutedwith approximately 10 mL of methanol. The fraction was then brought todryness under a stream of dry nitrogen. Using the HPLC method describedabove in Example 4, a small portion of the 20.2 mg of Peak B isolatedfor testing by MS and NMR was redissolved in mobile phase and injectedon the HPLC system to determine the purity of the fraction. Thisinjection of Peak B showed a purity of about 82% (FIG. 25).

Mass Spectral Analyses

Preliminary negative ion FAB-MS spectral data of the isolated fractionof Peak B indicated that the molecular weight was approximately 361 m/z(FIG. 26). A negative ion HR-MS study indicated a mass of 361.0744 amuthat compares well with the calculated mass of 361.0746 amu for theproposed molecular formula of C₁₈H₁₇O₆S₁ for Peak B.

Proton (¹H) and 2D COSY Nuclear Magnetic Resonance Spectroscopy

The ¹H-NMR and the 2D COSY spectra of Peak B in deuterated dimethylsulfoxide (d6-DMSO) are shown in FIGS. 27-29 and 30-32, respectively.The peak assignments, based upon the proton NMR spectra and COSYspectral couplings, are shown in Table 3 and are fully consistent withthe structure of Peak B.

Carbon (¹³C), 2D HMQC, and 2D HMBC Nuclear Magnetic Resonance

The ¹³C-NMR, HMQC, and HMBC spectra of Peak B in deuterated dimethylsulfoxide (d₆-DMSO) are shown in FIGS. 33-37, 38-41, and 42-47,respectively. In order to collect the data more quickly and with agreater signal to noise ratio, the ¹³C-NMR spectrum was obtainednon-quantitatively, and integrations were not performed. Peakassignments based upon the carbon NMR, HMQC, and HMBC spectralinterpretations are shown in Table 4, and are fully consistent with theproposed structure of Peak B. 2D HMBC data can be more difficult tointerpret, since it is possible that all crosspeaks are not observed.HMBC signals typically occur with H—C connectivities that are 2 to 4bonds removed, but also can detect some 1 to 2 bond connections.

TABLE 3 Summary Table of Proton NMR and COSY Band Assignments ChemicalShift Tenative Multi- Number Of COSY (ppm) plicty* Protons Couplings**Assignment 0.70 s 3 — 18  1.17 t 9 19  20  1.78 m 1 11b, 12b 12a 1.88 m1 14, 15b, 16b 15a 2.00 m 1 11a, 12a 12b 2.32 m 1 16b 16a 2.39 m 1 14,15a 15b 2.50 — — — solvent-DMSO 2.62 m 1 15a, 16a 16b 3.08 m 1 12b 11a3.10 q 6 20  19  3.12 m 1 12a 11b 3.14 d 1 15a, 15b 14  3.18 — — —solvent-MeOH 3.34 — — — solvent-H₂O 4.10 — — — solvent-MeOH 6.69 s 1 — 77.33 d of d 1 1, 4 2 7.77 d 1 2 1 7.89 d(w) 1 2 4 8.88 bs 1 — NH⁺ 9.83 s1 — 6(OH) *s—singlet, d—doublet, t—triplet, q—quartet, m—multiplet,b—broad, w—weak **weaker couplings are underlined

TABLE 4 Summary Table of Carbon NMR, HMQC, and HMBC Peak AssignmentsChemical Shift Tenative Number Of HMQC HMBC (ppm) Carbons CouplingsCouplings Assignments 8.6 3 1.17 — 20 12.7 1 0.70 12, 13, 14, 17 18 21.41 1.88, 2.39 13, 14, 17 15 22.9 1 3.08, 3.12 8, 9, 12, 13 11 28.8 11.78, 2.00 9, 11, 13, 14 12 36.1 1 2.32, 2.62 14, 15, 17 16 39-40 — — —solvent-DMSO 45.7 1 3.10 — 19 45.9 1 3.14 8, 9, 12, 13, 15, 14 18 46.9 3— — 13 48.5 — — — solvent-MeOH 106.0 1 6.69 4, 5, 6, 9, 14 7 111.7 17.89 2, 3, 6, 10 4 120.1 1 — — 9 121.8 1 7.33 3, 4, 10 2 123.7 1 7.77 3,4, 5, 6, 9, 10 1 124.2 1 — — 5 129.4 1 — — 10 133.3 1 — — 8 149.7 1 — —3 151.2 1 — — 6 218.9 1 — — 17 — — 9.83 5, 6, 7, 10 6(OH)ID and 2D NMR Spectral Interpretation

Peak B is a derivative of equilenin, which contains five aromaticprotons and ten aliphatic protons. The ¹H-NMR spectrum exhibits the tenexpected aliphatic protons, but only four main signals were observed inthe aromatic region (6.5-8.0 ppm) of the ¹H-NMR (FIG. 29) thatcorresponded to a 1:1:1:1 ratio. Based upon the splitting expected fromthe proposed structure for Peak B, these signals were consistent with anequilenin based ring structure substituted at one of the aromaticprotons. The four aromatic ¹H-NMR signals showed a strong singlet and astrong doublet, and a second singlet and doublet, which are weakly splitinto doublets.

Substitution at each of the possible aromatic positions would create adistinct splitting pattern. Substitution at the 1-position would createa single pair of strong doublets (H6 and H7) with a strong COSYcorrelation and a pair of singlets (H4 and H2) which would exhibit aweak COSY correlation and be weakly split by each other. Substitution atthe 2-position would create but a single pair of strong doublets (H6 andH7) with a strong COSY correlation and a pair of singlets (H4 and H1).Substitution of the aromatic ring system at the 4-position would createa pattern of 2 strong pairs of doublets with strong COSY correlations inthe spectrum. Substitution at the 6- or 7-position would create but asingle pair of strong doublets (H1 and H2) with a strong COSYcorrelation and a pair of singlets (H4 and H6 or H7). The H4 protonwould be expected to interact weakly with the H2 proton exhibiting aweak COSY correlation and causing the H2 doublet and the H4 singlet tobe weakly split by each other.

Based upon the splitting pattern of the aromatic protons, substitutionof the hydroxyl group in Peak B must be at either the H6 or H7 position.The pair of doublets at 7.77 and 7.33 ppm for H1 and H2, respectively,is shown to be adjacent from the 2D COSY spectrum (FIG. 32). H2 and H4at 7.89 ppm exhibited a weak COSY correlation that caused H2 to appearas a doublet of doublets due to splitting by both H1 and H4; and H4 as astrong singlet weakly split to a doublet. The assignments of thecorresponding carbons C1, C2, and C4 were based upon the HMQC spectra(FIG. 41) at 123.7, 121.8, and 111.7 ppm, respectively. The otheraromatic ring contains only one proton at 6.69 ppm for H6 or H7, whichwas observed as a singlet as expected since no other protons are nearbyto cause splitting. The corresponding carbon signal was assigned fromthe HMQC correlations at 106.0 ppm (FIG. 41).

In the ¹³C-NMR aromatic region (100-170 ppm) (FIG. 36), there were fourlarge signals and six smaller signals. Protonated carbons typically havelarger signals than non-protonated carbons and that was used indifferentiating among the aromatic carbon atoms. This was verified byobservation of only four HMQC signals in this region, which occur onlyfor carbons with directly attached protons, in the aromatic region (FIG.41). The remaining six aromatic carbon signals did not have HMQC peaks,and are therefore, non-protonated.

Two of the six non-protonated signals are shifted downfield to about 150ppm (149.7 and 151.2 ppm), which is typical of aromatic carbon atomsattached to an oxygen atom. This fits the proposed structure with thenormal 3-position hydroxy sulfate ester and the proposed hydroxylsubstitution on an aromatic position. The HMBC spectrum (FIG. 46) showsstrong correlations of the carbon signals at 149.7 ppm to H1 and H4 andthe carbon signal at 151.2 ppm to H4 and either H6 or H7. Based uponthose correlations the signal at 149.7 ppm must be C3. The signal at151.2 ppm must be C6 or the H4 correlation would have been a weak 4-bondcorrelation. Thus, the substitution is at the 6-position and thearomatic proton at 6.69 ppm and the aromatic carbon at 106.0 ppm areassigned H7 and C7, respectively. The remaining four non-protonatedcarbon atoms (120.1, 124.2, 129.4, and 133.3 ppm) match the number ofbridging non-protonated carbon atoms expected for the proposedstructure. Assignment of these four signals can be made from HMBCcorrelations (FIG. 46). H2 showed correlations to the carbon signals at111.7 (C4), 129.4, and 149.7 (C3) ppm. C10 was the only bridging carbonatom within 3 bonds of H2 and was assigned to the carbon signal at 129.4ppm. H1 exhibits correlations at 111.7 (C4), 120.1, 124.2, 129.4 (C10),149.7 (C3), and 151.2 (C6) ppm. H4 exhibits HMBC correlations to 121.8(02), 129.4 (C10), 149.7 (C3), and 151.2 (C6) ppm. H7 exhibits HMBCcorrelations to aromatic signals at 111.7 (C4), 120.1, 124.2, and 151.2(C6) ppm. Based upon these correlations, the carbon signals at 120.1 and124.2 ppm must correspond to C5 and C9, but their exact assignments arenot yet established.

There is a single strong signal downfield in the ¹H-NMR spectrum at 9.83ppm (FIG. 29). This region is typical of aromatic phenolic protons andthis signal is assigned as H6(OH). HMBC spectrum for this protonexhibits correlations at 106.0 (C7), 124.2, 129.4 (C10), and 151.2 (C6)ppm (FIG. 46). Based upon these correlations and the relationships ofthe other protons the signal at 124.2 ppm must be C5 and thus the signalat 120.1 ppm must be C9. This leaves only the aromatic carbon signal at133.3 ppm unassigned. Thus, the remaining aromatic signal by process ofelimination was assigned as C8.

The methyl region (0.5 to 1.5 ppm) of the ¹H-NMR spectrum (FIG. 28)shows a strong methyl signal split into a triplet at 1.17 ppm that isindicative of the methyl proton (H20) of the triethyl ammonium cation.This signal shows a strong COSY correlation to the quartet signal at3.10 ppm for the protons (H19) of the methylene group (FIG. 31). TheHMQC correlation spectrum (FIG. 39) showed corresponding carbon atoms at8.6 and 45.7 ppm for C20 and C19, respectively. The amine proton (NH⁺)of the cation was expected to have a ¹H-NMR chemical shift of about 8.0to 9.5 ppm; however, amines have the problem of slow exchange and oftenare not seen, or are only seen as a small broad peak in this region. TheNH proton in the ¹H-NMR spectrum was observed as a single broad signalat about 8.88 ppm for this compound (FIG. 29).

In the ¹³C-NMR aliphatic region (0-100 ppm) (FIGS. 34-35), there weretwo strong signals at 8.6 and 45.7 ppm for the triethyl ammonium cation,and seven signals for the aliphatic carbons of Peak B. The proposedstructure for Peak B contains seven aliphatic carbon atoms and a ketonecarbon. The seven aliphatic carbon signals were observed at 12.7, 21.4,22.9, 28.8, 36.1, 45.9, and 46.9 ppm. Carbonyl carbon atoms are known toshift far downfield to above 200 ppm. FIG. 37 showed such a signalpresent at 218.9 ppm and was assigned C17. Six of the seven signals showHMQC correlations to proton signals (FIGS. 39-40). Only the carbonsignal at 46.9 ppm did not exhibit a correlation to any proton signaland was considered a bridging carbon. The proposed structure has onebridging aliphatic carbon atom, and thus the peak at 46.9 ppm wasassigned as C13. The carbon signals at 12.7 and 45.9 ppm each correlatedto a single proton signal, whereas the other four carbon signalsobserved at 21.4, 22.9, 28.8, and 36.1 ppm each correlated to two protonsignals. This is often the case in saturated aliphatic ring systemssince the two protons of the methylene groups are present in differingelectronic environments and thus, exhibit different chemical shifts.

Inspection of the two carbon signals with a single proton HMQCcorrelation shows that the signal at 12.7 ppm is in the expected methylregion for ¹³C-NMR and correlates by HMQC to the proton signal at 0.70ppm. These protons at 0.70 ppm exhibited the expected integration ratiofor a methyl group of 3:1 relative to the individual aromatic protonsand are assigned as H18 based on the chemical shift and the HMQCcorrelations and is the only methyl group in the proposed structure forPeak B. The remaining carbon atom with one proton was observed at 45.9ppm was assigned as C14. The proton signal at 3.14 ppm correlated to itby HMQC and was assigned H14.

The remaining four aliphatic carbon signals each exhibited two HMQCcorrelations to proton signals. The carbon signal at 22.9 ppm correlatesto the protons at 3.08 and 3.12 ppm. The COSY spectrum of these twoprotons show that they couple to the proton signals at 1.78 and 2.00 ppmindicating the two sets are adjacent. The HMQC spectrum shows theseprotons both correlate to the carbon signal at 28.8 ppm. The carbonsignal at 21.4 ppm correlates to the protons at 1.88 and 2.39 ppm. TheCOSY spectrum of these two protons show that they couple to the protonsignals at 2.32 and 2.62 ppm, indicating the two sets are adjacent. TheHMQC spectrum shows these protons both correlate to the carbon signal at36.1 ppm. These observations are consistent with the proposed structureof Peak B that has two sets of adjacent methylene groups at C11 and C12,and at C15 and C16.

HMBC couplings (FIGS. 43-44) can be used to ascertain the identity andposition of each of the four methylene groups. The bridging carbon at120.1 (09) ppm correlates to only the protons of the carbon signals at22.9 and 28.8 ppm (FIG. 44). This observation verifies that the carbonsignals at 22.9 and 28.8 ppm must be assigned as C11 and C12 and thatthe other two carbon signals at 21.4 and 36.1 ppm must be assigned asC15 and C16, but their exact assignments are not yet established. Theproton at 0.70 (H18) ppm shows a HMBC correlation to the carbon signalsat 28.8, 45.9 (014), 46.9 (C13), and 218.9 (C17) ppm (FIG. 43). Basedupon this, the carbon signal at 28.8 ppm was assigned as C12 and thus,the carbon signal at 22.9 ppm must be C11. Based upon the HMQCcorrelations (FIG. 40) of each of these carbon signals, the protonsignals at 3.08 and 3.12 ppm can be assigned as H11a and H11b,respectively, and the protons at 1.78 and 2.00 ppm can be assigned asH12a and H12b, respectively. The proton at 3.14 (014) ppm shows COSYcorrelations to the protons at 1.88 and 2.39 ppm (FIG. 31). Based uponthese COSY correlations of the proton signal at 2.14 ppm, it must beadjacent to H14 and thus the carbon signal at 21.4 ppm must be C5 andthe carbon signal at 36.1 ppm must be C16. Based upon the HMQCcorrelations (FIG. 40) of each of the carbon signals, the proton signalsat 1.88 and 2.39 ppm can be assigned as H15a and H15b, respectively, andthe protons at 2.32 and 2.62 ppm can be assigned as H16a and H16b,respectively. Other HMBC correlations are detailed in Table 4 above andare consistent with the proposed structure of Peak B.

Additional signals in the proton NMR spectrum (FIGS. 28-29) wereobserved from water at 3.34 ppm, DMSO at 2.50 ppm, a small amount ofmethanol at 3.18 and 4.10 ppm, and other assorted small, unidentifiedaromatic and aliphatic “impurity” signals. In addition to the expectedaromatic and aliphatic signals of Peak B (FIG. 38), methanol wasobserved at 48.5 ppm (HMQC peak at 3.18 ppm).

Based on the observed NMR data the structure of Peak B corresponds wellto the proposed structure.

The present invention has been described herein with reference to itspreferred embodiments. The embodiments do not serve to limit theinvention, but are set forth for illustrative purposes. The scope of theinvention is defined by the claims that follow.

1. A compound having the following structure:

or pharmaceutically acceptable salts thereof wherein: R and R₁, are eachindependently hydrogen, sulfate or glucuronide and said compound has thefollowing physicochemical properties: (a) a peak located at about 1.2ppm in a ¹H-NMR spectrum; and (b) a peak located at about 45 ppm in a¹³C-NMR spectrum.
 2. The compound of claim 1, wherein the compound is acalcium salt thereof.
 3. The compound of claim 2, wherein the calciumsalt is selected from the group consisting of calcium citrate, calciumlactate, calcium fumurate, calcium acetate, calcium glycerophosphate,calcium chloride, calcium phosphate, calcium sulphate and calciumnitrate.
 4. The compound of claim 1, wherein the compound is greaterthan about 95% pure.
 5. A method of treating a subject in need ofestrogen therapy, said method comprising administering an effectiveamount of a compound having the following structure:

or pharmaceutically acceptable salts thereof wherein: R and R₁ are eachindependently hydrogen, sulfate or glucuronide and said compound has thefollowing physicochemical properties: (a) a peak located at about 1.2ppm in a ¹H-NMR spectrum; and (b) a peak located at about 45 ppm in a¹³C-NMR spectrum.
 6. The method of claim 5, wherein the compound orpharmaceutically acceptable salt is administered as a vaginal cream,gel, suppository or transdermal patch.
 7. The method of claim 5, whereinthe compound or pharmaceutically acceptable salt thereof is administeredas part of a pharmaceutical composition, said composition furthercomprising at least one additional pharmaceutically active ingredient.8. The method of claim 7, wherein the at least one additionalpharmaceutically active ingredient is selected from the group consistingof estrogenic compounds, androgenic compounds, progestin compounds,vasodilation agents, calcium salts, and vitamin D, and mixtures andcombinations thereof.
 9. The method of claim 5, wherein the conditiontreatable by estrogen therapy is selected from the group consisting ofvasomotor symptoms, atrophic vaginitis, osteoporosis, hypoestrogenismdue to hypogonadism, hypoestrogenism due to castration, hypoestrogenismdue to primary ovarian failure, breast cancer in selected persons withmetastatic disease, advanced androgen-dependent carcinoma of theprostate, abnormal uterine bleeding, and kraurosis vulvae.